title: Use of a high-throughput screen to identify Leptospira mutants unable to colonize the carrier host or cause disease in the acute model of infection.
abstract: The molecular basis for leptospirosis infection and colonization remains poorly understood, with no efficient methods available for screening libraries of mutants for attenuation. We analysed the attenuation of leptospiral transposon mutants in vivo using a high-throughput method by infecting animals with pooled sets of transposon mutants. A total of 95 mutants was analysed by this method in the hamster model of acute infection, and one mutant was identified as attenuated (M1233, lb058 mutant). All virulence factors identified in Leptospira to date have been characterized in the acute model of infection, neglecting the carrier host. To address this, a BALB/c mouse colonization model was established. The lb058 mutant and two mutants defective in LPS synthesis were colonization deficient in the mouse model. By applying the high-throughput screening method, a further five colonization-deficient mutants were identified for the mouse model; these included two mutants in genes encoding proteins with a predicted role in iron uptake (LB191/HbpA and LB194). Two attenuated mutants had transposon insertions in either la0589 or la2786 (encoding proteins of unknown function). The final attenuated mutant had an unexpected deletion of genes la0969-la0975 at the point of transposon insertion. This is the first description of defined, colonization-deficient mutants in a carrier host for Leptospira. These mutants were either not attenuated or only weakly attenuated in the hamster model of acute leptospirosis, thus illustrating that different factors that may be required in the carrier and acute models of leptospiral infection. High-throughput screening can reduce the number of animals used in virulence studies and increase the capacity to screen mutants for attenuation, thereby enhancing the likelihood of detecting unique virulence factors. A comparison of virulence factors required in the carrier and acute models of infection will help to unravel colonization and dissemination mechanisms of leptospirosis.
authors.affiliation: Department of Microbiology, Monash University, Wellington Road, Clayton, Victoria 3800, Australia.
authors.forename: Renee A
authors.initials: RA
authors.lastname: Marcsisin
authors.affiliation: Melioidosis Research Center, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand.
authors.forename: Thanatchaporn
authors.initials: T
authors.lastname: Bartpho
authors.affiliation: Victorian Bioinformatics Consortium, Monash University, Clayton, Victoria 3800, Australia.
authors.forename: Dieter M
authors.initials: DM
authors.lastname: Bulach
authors.affiliation: Section of Food Technology, Faculty of Natural Resources and Agro-Industry, Kasetsart University Chalermphrakiate Sakon Nakhon Province Campus, Sakon Nakhon 47000, Thailand.
authors.forename: Amporn
authors.initials: A
authors.lastname: Srikram
authors.affiliation: Department of Biochemistry, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand.
authors.forename: Rasana W
authors.initials: RW
authors.lastname: Sermswan
authors.affiliation: Australian Research Council Centre of Excellence in Structural and Functional Microbial Genomics, Department of Microbiology, Monash University, Clayton, Victoria 3800, Australia.|Victorian Bioinformatics Consortium, Monash University, Clayton, Victoria 3800, Australia.|Department of Microbiology, Monash University, Wellington Road, Clayton, Victoria 3800, Australia.
authors.forename: Ben
authors.initials: B
authors.lastname: Adler
authors.affiliation: Australian Research Council Centre of Excellence in Structural and Functional Microbial Genomics, Department of Microbiology, Monash University, Clayton, Victoria 3800, Australia.|Department of Microbiology, Monash University, Wellington Road, Clayton, Victoria 3800, Australia.
authors.forename: Gerald L
authors.initials: GL
authors.lastname: Murray
mesh_terms: D000818:Animals; D002353:Carrier State / Q000382:microbiology*; D004251:DNA Transposable Elements; D004195:Disease Models, Animal; D005260:Female; D055786:Gene Knockout Techniques; D005820:Genetic Testing; D057166:High-Throughput Screening Assays; D007919:Leptospira / Q000235:genetics / Q000472:pathogenicity*; D007922:Leptospirosis / Q000382:microbiology*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D016254:Mutagenesis, Insertional; D037521:Virulence Factors / Q000172:deficiency
chemical_list: D004251:DNA Transposable Elements; D037521:Virulence Factors